Plasmodium vivax recombinant CS protein vaccine Plasmodium vivax (Pv) is responsible for hundreds of millions of malaria cases annually, as many cases of malaria in travelers as P. falciparum (Pf), and a substantial economic burden. Severe malaria with mortality due to Pv has recently been reported from Oceania, south Asia, and South America. During the last decade, drug resistant Pv has emerged. There is a huge potential market for a Pv vaccine in travelers and military from the developed world, and among populations in countries with endemic Pv. Pv vaccine research has been neglected. The fact that Pf and Pv co-exist in most malaria endemic areas worldwide presents technical and ethical constraints for deployment of a vaccine effective only against Pf. Malaria cannot be eradicated without eliminating Pv. The only subunit malaria vaccines that have been reproducibly shown to prevent Pf malaria in humans are based on the Pf circumsporozoite protein (PfCSP). These protective vaccines elicit antibodies against the central repeat region of the molecule, which is conserved in all isolates of Pf, and against T cell epitopes in the C-terminal flanking region. It has not been possible to produce successfully a PfCSP recombinant (rec) protein vaccine that includes the N-terminal flanking region. Development of a PvCSP rec protein vaccine has been complicated by the fact that there are 2 major alleles of the PvCSP (210 and 247) based on variation in sequence of the central repeats. In Phase I we constructed PvCSP rec proteins that combined 3 copies of PvCSP 210 repeats and 3 copies of PvCSP 247 repeats with N-terminus (Protein 1), C- terminus (Protein 2) or N- and C-termini (Protein 3) of the PvCSP. All were expressed at levels adequate for GMP production. Protein 2 aggregated making purification and characterization difficult. All induced in mice antibodies that recognized PvCSP and Pv sporozoites expressing PvCSP 210 or 247, and inhibited Pv sporozoite invasion and development in human hepatocytes. Protein 2 did not induce antibodies against the Pv247 repeats, and could not be evaluated in T cell studies. Protein 3 induced better immune responses than Protein 1, and since Protein 3 includes essentially the full-length protein with all B and T cell epitopes, we have selected Protein 3 for Phase II. In Phase II we will: 1) Optimize protein 3 expression and generate a master clone and master cell bank, 2) Define specifications for fermentation procedure at scale under cGMPs, 3) Formulate candidate protein with 4 different adjuvants, 4) Down select optimal protein/adjuvant/formulation by comparative immunogenicity in mice and monkeys, 5) Define specifications for process, generate technical transfer records and documentation for cGMP manufacture, 6) Manufacture vaccine, conduct release assays, release bulk drug substance and conduct stability studies. By end of Phase II PvCSP Protein 3 vaccine/adjuvant will be available for pre-clinical toxicology studies and Phase 1/2a clinical trials. PUBLIC HEALTH RELEVANCE: Plasmodium vivax (Pv) is responsible for hundreds of millions of malaria cases annually, as many cases of malaria in travelers as P. falciparum (Pf), and a substantial economic burden. Severe malaria with mortality due to Pv has recently been reported from Oceania, south Asia, and South America. During the last decade, drug resistant Pv has emerged. There is a huge potential market for a Pv vaccine in travelers and military from the developed world, and among populations in countries with endemic Pv. This project will develop and manufacture such a vaccine.